Identification of the Sin3-binding site in Ume6 defines a two-step processfor conversion of Ume6 from a transcriptional repressor to an activator inyeast

The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction,vith the Sin3 corepressor a nd Rpd3 histone deacetylase and the meiotic activator Ime1. Here we show th at fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indic ating that an additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to activate lie in a pred icted amphipathic alpha helix and specifically disrupt interaction with Sin 3 but not with Teal, an activator of Ty transcription also found to interac t with Ume6 in a two-hybrid screen. The mutations cause a loss of repressio n by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we sho w interacts with the paired amphipathic helix 2 region of Sin3. Analysis of these mutants indicates that conversion of Ume6 to an activator involves t wo genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6. The mutants further demonstrate t hat premature expression and lack of subsequent rerepression of Ume6-Sin3-r egulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6 The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad -Max system and nuclear hormone receptors, and provides new insights into t he control of transcriptional repression and activation by the Ume6-URS1 re gulatory complex in yeast.